Development and Characterization of a Novel Fusion Protein Composed

Home , Protein G

J. Biochem. 133, 59-66 (2003) DOI : 10.1093/jb/mvg005 Development and Characterization of a Novel Fusion Protein Composed of a Human IgG1 Heavy Chain Constant Region and a Single-Chain Fragment Variable Antibody against Venezuelan Equine Encephalitis Virus

Wei-Gang Hu1,2, Azhar Z. Alvi1, Damon Chau1 , Jeffrey E. Coles1, R. Elaine Fulton1, M avanur R. Suresh2 and Les P. Nagata*,1

1Chemical Biological Defence Section, Defence Research & Development Canada -Suffield, Box 4000, Station Main, Medicine Hat, Alberta, Canada TZA 8K6; and 'Faculty of Pharmacy and Pharmaceutical Sciences, University of Alb erta, Edmonton, Alberta, Canada T6G 2N8

Received August 28, 2002; accepted October 25 , 2002

Murine monoclonal antibody 1A4A1 has been shown to recognize a conserved neu tralizing epitope of envelope glycoprotein E2 of Venezuelan equine encephalitis virus. It is a potential candidate for development of a second generation antibody for both immunodiagnosis and immunotherapy . In order to minimize the immunogenic ity of murine antibodies and to confer human immune effector functions on murine antibodies, a recombinant gene fusion was constructed . It encoded a human IgGl heavy chain constant region and a single-chain fragment variable antibody of 1A4A1 . After expression in bacteria as inclusion bodies, the recombinant antibody was puri fied and refolded in vitro. The recombinant soluble antibody was demonstrated to retain high antigen-binding affinity to Venezuelan equine encephalitis virus and to possess some human IgG crystallizable fragment domain functions, such as recogni tion by protein G and human complement Clq binding. On non-reducing and reduc ing gel electrophoresis analysis of proteolytic fragments of the recombinant antbody , di sulfide bond formation was found in the hinge region of the antibody. From these data, it was concluded that the recombinant antibody was capable of antigen recogni tion, and retained several functional activities. This work forms the basis for charac terization of the recombinant antibody as to efficacy in vivo.

Key words: antibody engineering, characterization, chimeric antibody, Venezuelan equine encephalitis virus.

Abbreviations: Ab, antibody; ABTS, 2,2•Œ-azino-di-(3-ethyl-benzthiazoline-sulfonic acid) diammonium salt; ADCC , Ab -dependent cellular cytotoxicity; CH , heavy chain constant region; ELISA, enzyme-linked immunosorbent assay; Fab, antigen-binding fragment; (Fab•Œ)2, fragment composed of two Fab units and a hinge region; Fe , crystalli zable fragment; HRP, horseradish peroxidase; IMAC, immobilized metal affinity chromatography; IPTG , isopro pyl ƒÀ-D-thiogalactopyranoside; MAb, monoclonal antibody; NTA, nitrilotriacetic acid; PBS, phosphate-buffered saline; PBST, PBS containing 0.1% Tween-20; ScFv, single-chain fragment variable; VEE , Venezuelan equine encephalitis virus; VL, light chain variable region; VH, heavy chain variable region; WEE , Western equine encephalitis virus.

Venezuelan equine encephalitis virus (VEE), a member of against VEE have been developed, these products are far the alphavirus genus of the family Togaviridae, is an from satisfactory. Approximately 20% of live-attenuated important pathogen for epidemics in humans, and epi TC-83 vaccine recipients fail to develop neutralizing anti zootics in rodents, horses, donkeys, and mules in the bodies (Abs), while another 20% exhibit reactogenicity Americas (1). VEE causes a spectrum of human diseases (5). A formaldehyde-inactivated vaccine, C-84, is well tol ranging from inapparent infection to acute encephalitis erated, but requires multiple immunization and periodic (2). Neurological disease appears in four to 14% of cases booster, and fails to provide protection against aerosol (3). The incidence of human infection during equine epi challenge in some rodent models (6). zootics could be up to 30% (4). VEE is a potential biologi VEE complexes comprise a group of antigenically cal warfare agent of concern, due to its high infectivity related, but distinct viruses divided into six subtypes (7) rate or aerosol transmissibility. However, there are no . VEE virions are composed of an icosahedral nucleocap antiviral drugs available that are effective against VEE. sid, which is surrounded by a lipid envelope containing Although live-attenuated and inactivated vaccines two structural glycoproteins, El and E2 (8) . Epitopes on El and E2 are the targets of neutralizing Abs . Studies have shown that the viral neutralizing epitopes are *T o whom correspondence should be addressed. Tel: +1-403-544 mainly located on the E2 protein, and that the E2c 4628, Fax: +1-403-544-3388, E-mail: [email protected] epitope appears to be the hub of the neutralization

Vol. 133, No. 1, 2003 59 © 2003 The Japanese Biochemical Society . 60 W.-G. Hu et al. epitopes (9). Monoclonal antibody (MAb) 1A1A4 is spe cific for Etc. This MAb has been shown to be efficient in protecting animals from a lethal peripheral challenge with virulent VEE (9). Murine MAbs, however, have serious disadvantages as therapeutic agents in humans (10, 11). They induce human anti-mouse Ab responses. Re-treatment may result in rapid clearance of the murine MAbs and ana phylaxis. Limitations to the use of murine MAbs for clin ical applications led to the development of single-chain fragment variable (scFv) Abs (12). ScFv Abs have several advantages compared with the mouse parental MAbs from which they are generated. They generally retain the same specificity and similar affinity to antigens (13), and exhibit decreased immunogenicity as compared with the parental mouse MAbs (14). Furthermore, scFv Abs can be produced economically and in a short time in bacteria or yeast (15, 16), and can be manipulated by genetic engi neering to form novel proteins by fusion with other mole cules, such as metal-binding proteins (17), cytokines (18), toxins (19), and T cell receptors (20). Our laboratory has cloned and characterized several scFv Abs against genus alphavirus, VEE or Western equine encephalitis virus (WEE) (21-23). An anti-VEE Fig. 1. pRSmA116huFc construct. The mA116 scFv Ab gene A116 scFv Ab was cloned from 1A4A1 MAb (24). How was cloned into the SfiI and NotI sites of pRSlOB5huFc to replace ever, in vitro binding assays demonstrated that A116 the 10B5 scFv Ab gene as described under "MATERIALS AND scFv Ab had low binding affinity to VEE, as compared to METHODS." the parental MAb. Sequence analysis of A116 revealed that three bases were missing in conserved framework-1 of the light chain variable region (VL). PCR-based site mutagenesis (Alvi et al., submitted). The mA116 scFv Ab directed mutagenesis was used to reintroduce the three DNA fragment was separated from vector pCANTAB 5E by missing bases, resulting in a repaired A116 scFv Ab, des restriction enzyme digestion with Sfi I/Not I, followed by ignated mA116 scFv Ab (Alvi et al., submitted). This gel electrophoresis and purification with a gel extraction repaired scFv showed affinity to VEE comparable to that kit (Qiagen, Mississauga, ON). The fragment was sub of the parental 1A4A1 MAb. cloned into the SfiIlNotI site of the pRS10B5huFc expres Antiviral immunity is complex, with several factors sion vector (22), replacing the anti-WEE 10B5 scFv Ab being involved, such as the Ab response, cell-mediated gene. The fragment was inserted in-frame with the se immunity, and induction of interferon. The Ab response quence encoding 36 kDa human IgG1 CH, just before the to viruses includes not only neutralization of infectivity CHI domain. A start codon, followed by a fragment con for susceptible host cells, but also host effector functions taining a 6 His tag and X-press epitope, was added at the 5•Œ such as complement-mediated lysis of infected host cells end for ease of purification and detection. The construct, and opsonization. The host effector functions of Abs are pRSmA116huFc, was shown in Fig. 1. The recombinant attributable to the crystallizable fragment (Fc) regions plasmid DNA was transferred into E. coli BL-21 (DE3) (10). ScFv Abs lack a Fc region, and thus lack effector pLys S competent cells containing a genomic source of T7 functions. RNA polymerase under lacUV5 promoter control (Invitro

In this study, we have genetically joined a truncated gen, Carlsbad, CA). The recombinant plasmid, containing human IgGI heavy chain constant region (CH) to anti the mAll6 scFv Ab gene insert, was confirmed by restric VEE mA116 scFv Ab, resulting in clone mA116huFc Ab. tion digestion and DNA sequencing. Six primers for DNA The goal of this work was to confer some of the human sequencing were made with an Oligo 1,000 DNA synthe associated effector functions on the scFv Ab without in sizer (Beckman Instruments, Fullerton, CA), and were as creasing the immunogenicity in humans. The mA116huFc follows: SC-1 (5•Œ-CATCATCATCATCATCAT-3•Œ), SC-9 (5•Œ Ab gene was expressed in E. coli, and the resulting prod CAGCAGCCAACTCAGCTT-3•Œ), SC-13 (5•Œ-ACTCCTGACA uct purified refolded in vitro, and characterized. The TCCTGTCG-3•Œ), HL-2 (5•Œ-TCTAACGGGACCAGAGAC results revealed that mA116huFc Ab retained antigen AAC-3•Œ), HL-3 (5•Œ-GGTAAGTTCAGGGACAGG-3•Œ), and binding affinity to VEE and possessed some human Ig GI HU-4 (5•Œ-TGCAAGGCCAGTCAGGATGTG-3•Œ) . The se Fc domain functions, such as recognition by protein G quencing reactions were performed with a Big Dye Termi and human Clq binding. natorTM cycle sequencing kit (PE Biosystems , Mississauga, ON) . The reaction products were purified on Centri-SepTM columns (Princeton Separations, Adelphia MATERIALS AND MATHODS , NJ), and then applied to an ABI 310 genetic analyzer system (Applied Bi Construction of pRSmA116huFc-The mA116 scFv Ab osystem, Fullerton, CA). Sequences were assembled and analyzed using Lasegene DNA software (DNA Star gene was originally derived from the A116 scFv Ab gene, , Madi which was subsequently replaced by site-directed son, WI).

J. Biochem. Chimeric Antibody against VEE 61

Expression, Purification, and Refolding of the Recom mercaptoethanol) or non-reducing conditions. The bands binant mA116huFc Ab-The recombinant protein ex were visualized by Coomassie Blue staining. In addition, pressed in E. coli as inclusion bodies was solubilized, mAll6-huFc Ab was digested with papain and pepsin purified, and refolded, as previously described, with (Sigma, Oakville, ON), respectively, by the method of minor modifications (22). Briefly, after induction for 3 h Andrew and Titus (25). Proteolytic fragments of the Ab with isopropyl ƒÀ-D-thiogalactopyranoside (IPTG), E. coli were analyzed by the above-described SDS-PAGE method. transformants harboring the pRSmAll6huFc expression Enzyme-Linked Immunosorbent Assay (ELISA)-The plasmid were harvested by centrifugation. Each cell pellet reactivity of mA116huFc Ab to VEE antigen was deter was resuspended in 5 mM borate sodium, pH 9.3, and 4 mined by ELISA. Nunc MaxisorpTM flat-bottomed 96-well M urea, and then a cell lysate was prepared by sonication plates (Invitrogen, Burlington, ON) were coated over (three cycles of 10 s; amplitude, 10 ƒÊm; 15 s cooling on night at 4•Ž with live-attenuated, whole VEE (strain TC ice), using a MSE Soniprep 150-probe sonicator (Wolf 83) at a fixed concentration of 10 ƒÊg/ml or various concen Laboratories, Pocklington, UK). The sonicates were cen trations, 0.01-100 ƒÊg/ml, in carbonate bicarbonate buffer, trifuged (13,000 xg for 10 min), and the pellets were pH 9.6, containing 0.02% sodium azide. The plates were resuspended in 5 mM borate sodium, pH 9.3, 8 mM urea, washed five times with PBST and then blocked twice in and 100 mM sodium chloride. Purification of the recom 2% BSA for 1 h at 37•Ž. After 5 washes with PBST, the binant protein was performed on TalonTM metal affinity plates were incubated for 1 h at 37 •Ž with various con resin (Clontech, Palo Alto, CA). A solution comprising 5 centrations, 0.01-100 ƒÊg/ml, or a fixed concentration of mM borate sodium, pH 9.3, 8 M urea, and 100 mM 10 ƒÊg/ml of a purified Ab (mA116huFc Ab, 1A4A1 MAb, sodium chloride was used as the wash buffer. Bound frac or mA116 scFv Ab) diluted in PBST Following five tions were eluted with 10-250 mM imidazole, and then 1 washes with PBST, the plates were incubated for 1 h at M arginine (final concentration) was added as a cosol 37•Ž with HRP-conjugated Ab diluted 1:3,000 in PBST vent, to facilitate the correct folding of the protein mole [HRP-donkey anti-human Ig for detection of mA116huFc cules. The recombinant protein (-100 ƒÊg/ml) was refolded Ab, HRP-goat anti-mouse Ig for 1A4A1 MAb, or HRP via removal of 8 M urea by dialysis against 5 mM borate anti-E tag (Amersham Pharmacia Biotech) for mA116 sodium, pH 99.3, and 1 M arginine at 4•Ž with air oxida scFv Ab]. Finally, the plates were washed five times with tion for 70 h; the cosolvent was then removed by dialysis PBST and developed for 30 min at room temperature against 5 mM borate sodium, pH 9.3, at 4•Ž for 24 h. The with a substrate consisting of 2,2•Œ-azino-di-(3-ethyl-benz purity was checked by SDS-PAGE and Coomassie Bril thiazoline-sulfonic acid) diammonium salt (ABTS) and liant Blue R-250 (Bio-Rad Laboratories, Mississauga, hydrogen peroxidate (Kirkegaard and Perry Laborato ON) staining after concentration by ultrafiltration using ries, Gathersburg, MD). The reactions were examined as a CentriconTM YM-30 (Millipore Corp., Bedford, MA). to the absorbance at 405 nm with a microplate SDS-PAGE and Western Blot Analysis-Protein sam autoreader (Molecular Devices, Sunnyvale, CA). ples were electrophoresed on 10% SDS-PAGE gels, using Protein G Binding Assay-The assay for Protein G a Mini-PROTEAN II apparatus (Bio-Rad Laboratories). - binding of the purified recombinant mA116huFc Ab was The bands were visualized by Coomassie Blue staining. modified based on the immunoprecipitation protocol (26). The separated proteins were also transferred to Immu MA116huFc Ab, 1A4A1 MAb, or mA116 scFv Ab, was nobilon-P membranes (Millipore Corp) by use of a incubated with washed protein G agarose (Invitrogen) in Western blot semi-dry transfer apparatus (Bio-Rad Labo radioimmunoprecipitation buffer (50 mM Tris-HCl, pH ratories) with Towbin buffer (25 mM Tris-HCl, pH 8.3, 7.4, 150 mM sodium chloride, 0.1% SDS, and 1% Triton 192 mM glycine, and 20% methanol). Unreacted sites X-100). After 1 h incubation with rocking at room tem were blocked with blocking buffer [3% non-fat skim milk perature, the agarose was collected by centrifugation at in phosphate-buffered saline (PBS)]. Blots were washed 13,000 •~g for 1 min at room temperature. The pellet was three times for 5 min with PBS containing 0.1% Tween washed three times with radioimmunoprecipitation -20 (PBST). Some blots were then incubated for 1 h at buffer and then centrifuged at 13,000 •~g for 1 min. The room temperature with a 1:5,000 dilution of mouse anti protein G binding complexes were resuspended in lae - Xpress Ab (Invitrogen). These blots were then washed mmli sample buffer containing 5% 2-mercaptoethanol three times for 5 min with PBST and incubated for 1 hat and then heated in boiling water for 10 min. After centrif room temperature with a 1:3,000 dilution of horseradish ugation, the supernatants were run on a 10% SDS-PAGE peroxidase (HRP)-conjugated goat anti-mouse Ig (Caltag gel, followed by Coomassie Blue staining. Laboratories, Burlingame, CA) in PBST. Other blots were Human Complement Clq Binding Assay-The capacity incubated directly with either a 1:2,000 dilution of HRP of purified recombinant mA116huFc Ab to bind to human -conjugated donkey anti-human Ig (Jackson ImmunoRe complement Clq was assayed using a circulating search Laboratories, West Grove, PA) or a 1:1,000 dilu immune complex-Clq test kit (Quidel Corp., San Diego, tion of HRP-conjugated Ni-nitrilotriacetic acid (NTA) CA) in accordance with the manufacturer's instructions. (Qiagen) at room temperature for 1 h. After three washes In brief, eight-well strips coated with human Clq protein for 5 min with PBST, and 2 washes for 2 min with deion were rehydrated with wash buffer (0.05% Tween-20 and ized water, specific binding was detected with an 0.01% thimerosal in PBS). The purified recombinant enhanced chemilumeniscence kit (Amersham Pharmacia mA116huFc Ab, mA116 scFv Ab, a human IgGl (positive Biotech, Baie d'Urfe', QC). control) (Sigma), or a human IgG4 (negative control) Inter-Chain Disulfide Bond Formation Assay-MA116 (Sigma) was serially diluted from 15 nM with the wash huFc Ab, 1A4A1 MAb, and mA116 scFv Ab were electro buffer, and then incubated with the strips for 1 h at room phoresed on 10% SDS-PAGE gels under reducing (5% 2 temperature. After five washes with the wash buffer,

Vol. 133, No. 1, 2003 62 W-G. Hu et al.

Fig. 2. SDS-PAGE analysis of samples obtained on the purifi cation of mAll6huFc Ab. Samples were resolved on a 10% poly acrylamide gel and then stained with Coomassie Blue. Lane 1, molecular weight markers; 2, bacterial lysate; 3 solubilized protein fraction; 4, column flow-through fraction; 5, 10 and 20 mM imida zole eluates; 6, final protein preparation.

HRP-conjugated goat anti-human Ab was added to each well and the strips were incubated at room temperature for 30 min, after which they were washed five times with the wash buffer and then incubated with an ABTS sub strate solution for 30 min. To stop the enzymatic reac tion, a stop solution containing 250 mM oxalic acid was Fig. 3. Western blotting analysis of samples obtained on puri fication of mAll6huFc Ab. Samples were resolved by SDS added to the wells, and then the absorbance was meas PAGE, transferred to Immunobilon-P membranes, and then probed ured at 405 nm with the microplate autoreader. with (A) HRP-conjugated anti-human Ig, (B) HRP-conjugated Ni NTA, or (C) anti-Xpress Ab, followed by HRP-conjugated anti RESULTS mouse Ig. Lane 1, bacterial lysate; 2, solubilized protein fraction; 3, column flow-through fraction; 4, 10 and 20 mM imidazole eluates; 5, final protein preparation. Construction, Expression, and Purification-The pRSlOB5huFc gene construct, in which the anti-WEE virus 10B5 scFv Ab gene was linked with a truncated human IgG1 CH (22), and the anti-VEE mA116 scFv Ab that there was a large amount of protein in the bacterial lysate of molecular weight -70 kDa, corresponding to the gene in pCANTAB5E were used as source materials to create a mA116huFc Ab gene construct. The 10B5 scFv predicted size (68 kDa) of mA116huFc Ab (Fig. 2, lane 2). Ab gene of pRSlOB5huFc was replaced by the mA116 After centrifugation of the lysate, and dissolution of the scFv Ab gene. The resulting plasmid, designated as pellet with a denaturing agent, many of the proteins were removed from the lysate (Fig. 2, lane 3). The solubi pRSmA116huFc, contained the mA116 scFv Ab gene, arranged in a heavy chain variable region (VH)-VL orien lized protein fraction was incubated with metal affinity tation via a (Gly4Ser)3 linker. This was fused to a trun resin, and then loaded on to an empty column. After thor cated human IgGl CH under the control of the T7 pro ough washing, the bound fractions were eluted with an moter (Fig. 1). In addition, there was a sequence with a imidazole gradient (10 to 250 mM) in elution buffer. The molecular weight of 6 kDa, including a 6His tag for 10 and 20 mM imidazole eluates each gave a major band immobilized metal affinity chromatography (IMAC) puri at -70 kDa accompanied by some weak bands (Fig. 2, fication and a Xpress epitope for detection with anti lane 5), whereas for the eluates with imidazole concen Xpress Ab, fused upstream of the VH. The mA116 scFv trations of 50 mM and greater, only the 70 kDa band was Ab gene was 723 by in length, encoding 241 residues with observed (Fig. 2, lane 6). With this elution protocol, the a molecular weight of 26 kDa. The first 66 bases of the expressed protein could be purified to >90%. The yield of CH1 domain were missing from the human IgG1 CH, as purified recombinant mA116huFc Ab was about 30 mg the parental IgGl vector was originally designed for clon per liter of culture medium. ing CH domains from antigen-binding fragments (Fabs). Biochemical Characterization-To confirm the presence The encoded CH protein comprised 322 residues with a of intact, expressed mA116huFc Ab, a series of Western molecular weight of 36 kDa. The theoretical molecular blotting experiments was performed, in which the 70 kDa weight of the whole fusion protein, including the 6His tag protein was detected with HRP-conjugated anti-human and X-press epitope, was about 68 kDa. Ig, HRP-conjugated-Ni-NTA, and anti-Xpress epitope , f The mA116huFc Ab was expressed in E. coli BL-21 ollowed by HRP-conjugated-anti-mouse Ig, respectively . cells and purified by IMAC. SDS-PAGE demonstrated As shown in Fig. 3, the 70 kDa protein was recognized in

J. Biochem. Chimeric Antibody against VEE 63

Fig. 4. Disulfide bond formation assay. A: Abs were resolved by SDS-PAGE under reducing and non-reducing conditions, and then stained with Commassie Blue. Molecular weight markers (lane 1); mA116huFc Ab under reducing (lane 2) and non-reducing (lane 3) conditions; 1A4A1 MAb under reducing (lane 4) and non-reducing (lane 5) conditions; and mA116 scFv Ab under reducing (lane 6) and Fig. 5. VEE antigen binding assaying by ELISA. A: Various con non-reducing (lane 7) conditions. B: SDS-PAGE analysis of proteo lytic fragments of mAl l6huFc Ab digested with papain and pepsin, centrations of Abs were added to a 96-well plate coated with 10 ƒÊg/ ml of VEE. B: 10 ƒÊg/ml of Abs was added to a 96-well plate coated respectively. Molecular weight markers (lane 1); proteolytic frag with various concentrations of VEE. Binding was detected with ments of mAll6huFc Ab with papain under non-reducing (lane 2) HRP-conjugated anti-human Ig, anti-mouse Ig, and anti-E-tag Ab, and reducing (lane 3) conditions; and proteolytic fragments of followed by an ABTS solution. Each point represents the mean of mAll6huFc Ab with pepsin under non-reducing (lane 4) and reduc four determinations. ing (lane 5) conditions.

migrated as only one band corresponding to a molecular Western blots by all three probes. The HRP-conjugated weight of -30 kDa under both reducing and non-reducing Ni-NTA and anti-Xpress Ab also detected a 32 kDa frag conditions (Fig. 4A, lanes 6 and 7). ment in the crude fractions, however, this fragment was In order to confirm the inter-chain disulfide bond for not present in the purified fraction (Fig. 3, B and C, lane mation in the hinge region of mAl16huFc Ab, the Ab was 5). digested with papain and pepsin, respectively, and then The hinge region of an Ab is responsible for disulfide subjected to SDS-PAGE under non-reducing and reduc bond formation between two identical Ab heavy chains. ing conditions. As we know, papain can specifically cleave Since mA116huFc Ab contained an intact hinge region, on the amino-terminal side of a inter-chain disulfide inter-chain disulfide bond formation was examined by bridge in the hinge region of an IgG molecule, yielding a comparing the Ab protein under reducing and non-reduc monovalent Fab, the remaining Fc fragment (around 27 ing conditions on SDS-PAGE (Fig. 4A). Under reducing kDa) and some other smaller fragments (25). Pepsin conditions (addition of 5% 2-mercaptoethanol), 1A4A1 cleaves on the carboxyl-terminal side of the same bridge, MAb appeared as two bands, corresponding to molecular generating a bivalent fragment composed of two Fab weights of 50 and 25 kDa, representing heavy and light units plus the hinge region [(Fab•Œ)2], and some small Fe chains, respectively (Fig. 4A, lane 4). Under non-reducing fragments (25). As shown on Fig. 4B, papain-digested conditions, 1A4A1 MAb appeared as a high molecule mAll6huFc Ab appeared as a band corresponding to 40 weight aggregate (Fig. 4A, lane 5). Under reducing condi kDa, corresponding in size to monovalent mAll6scFv tions, mA116huFc Ab migrated as one band correspond plus a CH1 domain, accompanied by several small frag ing to a molecular weight of -70 kDa and, under non ments under both non-reducing and reducing conditions reducing conditions, as a high molecule weight aggregate (lanes 2 and 3). Pepsin-digested mA116huFc Ab gave a (Fig. 4A, lanes 2 and 3). As expected, mA116 scFv Ab major band corresponding to a molecular weight of -90

Vol. 133, No. 1, 2003 64 W-G. Hu et al.

Fig. 6. Protein G binding assay. Abs were incubated with protein G agarose and then precipitated by centrifugation. The pellets were Fig. 7. Clq binding assay. Various concentrations of Abs were subjected to SDS-PAGE with Coomassie Blue staining. Lane 1, added to strips coated with human Clq protein. Binding was molecular weight markers; 2, mAll6huFc Ab only; 3, 1A4A1 MAb detected with HRP-conjugated anti-human IgG, followed by an only; 4, mA116 scFv Ab only; 5, mAll6huFc Ab precipitated with ABTS solution. Each point represents the mean of four determina protein G; 6, 1A4A1 MAb precipitated with protein G; 7, mA116 tions. scFv Ab precipitated with protein G.

DISCUSSION kDa, corresponding in size to bivalent mA116 scFv plus a CH1 domain and hinge region, and a faint band of -45 The E. coli expression system has been commonly applied kDa, accompanied by several small fragments under non for recombinant immunoglobulin fragments since with reducing conditions (lane 4). But under reducing condi this system it is possible to produce proteins in large tions, only a band corresponding to -45 kDa was quantities (27). However, the expression of heterologous observed besides several small fragments (lane 5). proteins in bacteria has been hampered by the fact that Binding Properties to VEE Antigen-Initially, mA116 the large amount of protein produced in the reducing huFc Ab was confirmed to bind to E2 protein of VEE, con environment of the cytoplasm of bacteria leads to the for sistent with the specificity of the parental 1A4A1 MAb, mation of insoluble inclusion bodies, which contain on Western blotting (data not shown). The immunoreac unfolded protein. It is necessary to solubilize and then tivity of mAl16huFc Ab toward VEE antigen was further refold the protein into the structurally correct form examined by ELISA. When the plates were coated with a before it becomes functionally active. The inclusion bod fixed concentration of inactivated VEE (10 ƒÊg/ml), ies can be easily dissolved with denaturants, such as gua mA116huFc Ab bound to VEE in a dose-dependent man nidine-HCl or urea, and then can be used for the refold ner, similar to the binding to VEE of the parental 1A4A1 ing procedure, which promotes disulphide bond MAb and mA116 scFv Ab (Fig. 5A). An additional ELISA formation and adoption of the appropriate three-dimen test was performed in which a concentration gradient of sional shape (28). Disulphide bond formation can be VEE was titrated against a fixed concentration of Abs (10 induced by simple air oxidation (29) or a mixture of pg/ml). A similar dose-response relationship was reduced and oxidized thiol groups (30). Refolding can be observed (Fig. 5B). promoted by artificial chaperones (31) or a stabilizing co Protein G and Clq Binding-Although 22 residues solvent, such as arginine, which can facilitate the forma were missing from the CH1 domain of mA116huFc Ab, tion of the native structure of a protein during refolding the CH2 and CH3 domains were intact. The CH2 and (32). However, there is no universal refolding protocol CH3 domains are believed to be associated with essential suitable for every antibody or antibody fragment since features of CH of the Abs, such as binding to proteins G each of them is unique. In our study, we solubilized the and A, and retention of binding sites essential for effector inclusion bodies with the denaturant 8 M urea . After functions. To determine the affinity of binding of mA116 purification, we allowed disulphide bond formation huFc Ab to protein G, mAl16huFc Ab was incubated with through air oxidation and refolded mAll6huFc Ab in the protein G agarose and then, after thorough washing and presence of arginine. centrifugation, analyzed by SDS-PAGE. As showed in The results of Western blot analysis confirmed that the Fig. 6, mA116huFc Ab, like parental 1A4A1 MAb, effi refolded recombinant protein was intact , with a molecul ciently bound to protein G agarose, while mA116 scFv Ab ar weight of -70 kDa. Comparison of the SDS-PAGE did not. To determine whether mA116huFc Ab could ini electrophoretic patterns of mAll6huFc Ab obtained tiate complement activation, one of the important effec under reducing and non-reducing conditions was con tor functions, an ELISA was performed, in which human ducted. As expected, mA116huFc Ab migrated as a high Clq was coated on the strips. MA116huFc Ab like a molecular weight aggregate under non-reducing condi human IgG1 could efficiently bind to human Clq in a tions, as did the parental 1A4A1 MAb . Under reducing dose-dependent manner, while mA116 scFv Ab and a conditions, mA116huFc Ab migrated as one band corre human IgG4 did not bind to human Clq (Fig. 7). sponding to a molecular weight of -70 kDa , correspond

J Biochem. Chimeric Antibody against VEE 65 ing in size to the monovalent mA116huFc Ab. These could change its binding ability to complement from min results suggested that there was inter-chain disulfide imal to profound (36-39). However, our results indicated bond formation in the mA116huFc Ab. Proteolytic diges that there was little to no effect of aglycosylation on the tion of mA116huFc Ab with papain and pepsin, and anal ability of mA116huFc to bind to complement. ysis of the fragments by SDS-PAGE under both non There have been several reports on recombinant reducing and reducing conditions were performed. fusions of scFv and different parts of the Fe fragment of Papain-digested mA116huFc Ab gave a band correspond human IgG. Coloma and Morrison reported CH3-scFv ing to 40 kDa, matching that of the monovalent scFv plus had some associated effector functions, such as a long the CH1 domain under both non-reducing and reducing half-life and Fe receptor binding (40). Munoz et al. fused conditions, which probably excluded the possibility that a scFv to hinge-CH2-CH3 to confirm complement C3 there was any inter-chain disulfide bond in the region of could efficiently bind to the remaining constant domains scFv plus the CHI domain. On the other hand, a band as well as the CHI domain (41). Muller et al. showed that corresponding to -90 kDa of pepsin-digested mAl l6huFc a scFv fused to the CHI domain became linked to another Ab on SDS-PAGE under non-reducing conditions migrated scFv fused to CL of human IgG via a disulfide bond to as a band corresponding to -45 kDa under reducing con form bispecific miniantibodies (42). Gilliland et al. and ditions, suggesting the -90 kDa protein was a dimer, Shan et al. fused hinge-CH2-CH3 to a scFv simply for matching that of bivalent scFv plus the CH1 domain and easy purification using staphylococcal protein A (43). We hinge region. Since there was no inter-chain disulfide for fused CHl-hinge-CH2-CH3 to anti-VEE scFv in order to mation in scFv plus the CHI domain, inter-chain disul confer on the scFv some of the human-associated effector fide formation should have occurred in the hinge region functions without increasing the immunogenicity in of mA116huFc Ab. humans. This fusion Ab was shown to be a disulfide The in vitro binding characteristics of mA116huFc Ab linked homodimer, which demonstrated retention of anti to VEE antigen were assayed by ELISA. The mAl l6huFc gen-binding affinity to VEE antigen. In addition, this Ab Ab exhibited strong binding activity toward VEE, indi was shown to possess some human Ig G1 Fc domain func cating that the folding was appropriate for the formation tions, such as protein G and human Clq binding. From of antigen-binding sites. The parental 1A1A4 MAb and these findings, it was concluded that this Ab was in a mA116 scFv Ab showed similar binding activity to VEE, native, functionally active form. This work will form the however, direct comparison of the binding affinitties of the basis for further studies to characterize mA116huFc Ab three Abs was not possible by ELISA, since each Ab for efficacy studies in vivo. required a different conjugated secondary Ab. The results of the protein G binding assay indicated This study was supported by a Natural Sciences and Engi that mAll6huFc Ab could bind to protein G, thus sug neering Research Council and Department of National gesting that the constant region of mA116huFc Ab was Defence (NSERC-DND) partnership program. Dr. Wei-Gang correctly folded. Protein G specifically interacts with the Hu is the recipient of a research postdoctoral fellowship CH2 and CH3 domains of Ab (33). The finding that award from the Defence Research & Development Canada/ mA116huFc Ab could bind to protein G suggested that Canadian Institutes of Health Research. the Fc region must be close in structure to the native pro file. We were also interested in determining whether or REFERENCES not the constant region in mAl l6huFc Ab was folded well enough to bind to complement. The classical pathway of 1. Johnston, R.E. and Peters, C.J. (1996) Alphaviruses in Fields complement activation is initiated by the constant region virology (Fields, B.N., Knipe, D.M., and Honley, P.M., eds.) 3rd ed., pp. 843-898, Raven Publishers, Philadelphia, PA of an Ab binding to Clq, a constituent of the first compo 2. Franck, P.T. and Johnson, K.M. (1970) An outbreak of Venezue nent of complement (34). The abilities of human IgG sub lan encephalitis in man in the Panama Canal Zone. Am. J. types to bind to human Clq vary widely. IgG/ can effi Trop. Med. 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